Neural crest cells are a multipotent cell type that migrates extensively in vertebrate embryos to form the majority of the craniofacial skeleton, outflow tract of the heart, and peripheral nervous system. Common birth defects such as cleft lip and palate result from abnormalities in neural crest cell development. Despite its fundamental importance, the mechanisms that regulate neural crest cell migration and motility are unclear. The lysine methyltransferase nuclear receptor-binding SET domain-containing 3 (NSD3) was identified in a screen for genes upregulated as neural crest cells become migratory. Preliminary results indicate that methylation and NSD3 are required for neural crest development. My hypothesis is that nuclear and cytoplasmic protein methylation by NSD3 is required for neural crest cell specification and migration. Protein methylation is a previously unexplored regulatory mechanism for neural crest cell development. Aim 1 will examine the requirement for NSD3 in the neural crest. Preliminary data demonstrating an essential role for NSD3 in neural crest specification and migration will be extended by examining additional neural crest markers. The requirement for NSD3 methyltransferase activity will also be established. Aim 2 will characterize the role of NSD3-mediated methylation in regulating neural crest cell motility through time-lapse imaging on live, migratory neural crest cells. Migration rate and directionality, as well as particular motile behaviors such as membrane protrusion and adhesion, will be compared between control and NSD3 loss of function conditions. Finally, Aim 3 will target dominant negative NSD3 constructs to the nucleus or cytoplasm to specifically assess the importance of histone versus cytoplasmic non-histone protein methylation in the neural crest. Subcellular localization of NSD3 will also be imaged in live, motile cells with a fluorescently tagged NSD3 construct. Altogether, imaging and loss-of-function experiments will establish the essential role of NSD3-mediated protein methylation in neural crest development and identify particular motile behaviors regulated by NSD3, such as protrusion extension or adhesion formation. This research will suggest potential targets of NSD3 for future research and will provide fundamental insights into the mechanics of neural crest migration and formation of craniofacial structures. These findings will suggest avenues for the prevention and treatment for common neural crest-based disorders, such as cleft lip and palate.